Missense but mis-spliced: germline TP53 variant c.671A>C (p.E224A) and the path from uncertainty to pathogenicity

The TP53 gene encodes the well-known P53 tumor suppressor protein, which plays a crucial role in preventing cancer development. Germline TP53 variants cause Li-Fraumeni Syndrome (LFS), an autosomal dominant disorder associated with early-onset cancers, including breast cancer, brain tumors, leukemias, bone cancers, and soft tissue sarcomas. Functional studies in yeast and human cells demonstrated that TP53 variants can have various effects, such as partial or complete loss of function and even gain of pro-oncogenic activities.

Here, we identified a germline TP53 variant c.671A>C, resulting in the missense mutant protein p.E224A in the context of early-onset retroperitoneal rhabdomyosarcoma occurring in a child with a notable family history of cancer, suggestive of LFS. The variant was initially classified as a variant of uncertain significance (VUS). Functional assays in yeast and human cells demonstrated wild type-like activity of the protein p.E224A; however, in silico analysis predicted at RNA level a splicing defect, which we further investigated using a minigene approach. This analysis showed that the variant c.671A>C causes the skipping of exon 6, potentially introducing a frameshift in cDNA and a premature stop codon, which likely triggers nonsense-mediated mRNA decay; the loss of heterozygosity at the c.671 position in the parent’s TP53 transcript further confirmed the splicing impairment.

In summary, these findings supported reclassifying the TP53 germline variant c.671A>C (p.E224A) from VUS to likely pathogenic, providing a definitive molecular diagnosis for family counseling. Additionally, this study sheds light on how certain TP53 variants that are defined as missense, can be linked to disease mechanisms through RNA splicing disruption, highlighting the need for their deep functional assessment.